Impact of Lachancea thermotolerans on Chemical Composition and Sensory Profiles of Viognier Wines.

Viognier is a warm climate grape variety prone to loss of acidity and accumulation of excessive sugars. The yeast Lachancea thermotolerans can improve the stability and balance of such wines due to the partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in co-inoculations and sequential inoculations with Saccharomyces cerevisiae in high sugar/pH Viognier fermentations. The results highlighted the dichotomy between the non-acidified and the bio-acidified L. thermotolerans treatments, with either comparable or up to 0.5 units lower pH relative to the S. cerevisiae control. Significant differences were detected in a range of flavour-active yeast volatile metabolites. The perceived acidity mirrored the modulations in wine pH/TA, as confirmed via "Rate-All-That-Apply" sensory analysis. Despite major variations in the volatile composition and acidity alike, the varietal aromatic expression (i.e., stone fruit aroma/flavour) remained conserved between the treatments.

Isolation and Characterization of High-Ethanol-Tolerance Lactic Acid Bacteria from Australian Wine.

Lactic acid bacteria are very important in winemaking. In this study, 108 lactic acid bacteria isolates were obtained from high-ethanol-content (~17% (v/v)) Grenache wines during uninoculated malolactic fermentation (MLF). The 16S rRNA and species-specific PCR showed that 104 of these were Oenococcusoeni, three were Lactobacillus hilgardii, and one was Staphylococcus pasteuri. AFLP of HindIII and MseI digests of the genomic DNA of the O. oeni strains was developed for the first time to discriminate the strains. The results showed that the method was a suitable technique for discriminating the O. oeni strains. Based on the cluster analysis, nine O. oeni strains were chosen for inclusion in an ethanol tolerance assay involving monitoring of optical density (ABS600nm) and viable plating. Several O. oeni strains (G63, G46, G71, G39) survived and grew well in MRS-AJ with 17% (v/v) ethanol, while the commercial O. oeni reference strain did not. Strain G63 could also survive and grow for 168 h after inoculation in MRS-AJ medium with 19% (v/v) ethanol. These results suggest that O. oeni G63, G46, G71, and G39 could potentially be used as MLF starters for high-ethanol-content wines. All three L. hilgardii strains could survive and grow in MRS-AJ with 19% (v/v) ethanol, perhaps also indicating their suitability as next-generation MLF starter cultures.

Impact of Lachancea thermotolerans on chemical composition and sensory profiles of Merlot wines.

Wines from warm(ing) climates often contain excessive ethanol but lack acidity. The yeast Lachancea thermotolerans can ameliorate such wines due to partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in two inoculation modalities (sequential and co-inoculation) to Saccharomyces cerevisiae and un-inoculated treatments in high sugar/low acidity Merlot fermentations. The pH and ethanol levels in mixed-culture dry wines were either comparable, or significantly lower than in controls (decrease of up to 0.5 units and 0.90% v/v, respectively). The analysis of volatile compounds revealed marked differences in major flavour-active yeast metabolites, including up to a thirty-fold increase in ethyl lactate in certain L. thermotolerans modalities. The wines significantly differed in acidity perception, alongside 18 other sensory attributes. Together, these results highlight the potential of some L. thermotolerans strains to produce 'fresher' wines with lower ethanol content and improved flavour/balance.

Lower-alcohol wines produced by Metschnikowia pulcherrima and Saccharomyces cerevisiae co-fermentations: The effect of sequential inoculation timing.

In Latin, 'pulcherrima' is a superlative form of an adjective that translates as beautiful. Apart from being 'the most beautiful' yeast, Metschnikowia pulcherrima has a remarkable potential in production of wines with lower ethanol content. The oenological performance of six M. pulcherrima strains was hereby tested in sequential cultures with Saccharomyces cerevisiae. The best-performing strain MP2 was further characterised in fermentations with different S. cerevisiae inoculation delays in both white grape juice and Chemically Defined Grape Juice Medium (CDGJM). The analysis of main metabolites, undertaken prior to sequential inoculations and upon fermentation completion, highlighted metabolic interactions and carbon sinks other than ethanol in MP2 treatments. Depending on the inoculation delay, MP2 white wines contained between 0.6% and 1.2% (v/v) less ethanol than the S. cerevisiae monoculture, with even larger decreases detected in the CDGJM. The MP2 treatments also contained higher concentrations of TCA cycle by-products (i.e. fumarate and succinate) and glycerol, and lower concentrations of acetic acid. The analysis of volatile compounds showed increased production of acetate esters and higher alcohols in all MP2 wines, alongside other compositional alterations arising from the S. cerevisiae inoculation delay.

Evaluation of indigenous non-Saccharomyces yeasts isolated from a South Australian vineyard for their potential as wine starter cultures.

The use of non-Saccharomyces yeast in conjunction with Saccharomyces cerevisiae in wine fermentation is a growing trend in the wine industry. Non-Saccharomyces, through their distinctive production of secondary metabolites, have the potential to positively contribute to wine sensory profile. To discover new candidate strains for development as starter cultures, indigenous non-Saccharomyces were isolated from un-inoculated fermenting Shiraz musts from a South Australian vineyard (McLaren Vale wine region) and characterised. Among the 77 isolates, 7 species belonging to 5 genera (Kazachstania, Aureobasidium, Meyerozyma, Wickerhamomyces and Torulaspora) were identified by sequencing the internal transcribed spacer regions of the 5.8S rRNA gene (ITS1-5.8S-ITS2 region). The indigenous isolates were evaluated for oenological properties, namely, ethanol tolerance, enzyme activity, and H2S production. To determine their potential industrial use as starter cultures, representative isolates of each species were assessed in a sterile chemically defined grape juice and Viognier grape juice to evaluate their contribution to fermentation kinetics and production of key metabolites, including volatile compounds.

Mathematical modelling of anthocyanin mass transfer to predict extraction in simulated red wine fermentation scenarios.

Anthocyanins are polyphenolic compounds present in grapes that are responsible for the initial colour of red wine and their incorporation into derived pigments leads to long term colour stability. The ability to predict the effect of process variables, either controlled by winemakers or that naturally change throughout fermentation, on the extraction of anthocyanins is vital to producing red wine of high quality. A 23 factorial experiment with additional points located at central factor conditions was used to determine the impact of temperature, sugar and ethanol concentrations on the mass transfer properties of anthocyanins from fresh Pinot noir grape solids. Factor conditions were chosen to replicate ethanol and sugar concentrations that would be found in a 14% red wine and its respective unfermented juice. A previously described mathematical model was applied to anthocyanin extraction curves to determine mass transfer coefficients and distribution constants for the generation of response surface models able to predict these mass transfer variables for dynamic fermentation scenarios. The coefficient of determination for the model solution exceeded 0.94 in all cases, demonstrating a good agreement between experimental and mathematically-derived anthocyanin concentrations. Following this, simulations of anthocyanin extraction under fermentation conditions were conducted using a previously developed wine fermentation model allowing for the prediction of extraction rates and anthocyanin concentrations under various winemaking scenarios. The extraction simulations predicted a previously observed but so far undescribed anthocyanin extraction pattern during fermentation.

Competition experiments between Brettanomyces bruxellensis strains reveal specific adaptation to sulfur dioxide and complex interactions at intraspecies level.

Recent studies have suggested a strong niche adaptation for Brettanomyces bruxellensis strains according to human-related fermentation environments, including beer, wine and bioethanol. This is further supported by a correlation between B. bruxellensis genetic grouping and tolerance to SO2, the main antimicrobial used in wine. The allotriploid AWRI1499-like cluster, in particular, shows high SO2 tolerance suggesting that the genetic configuration observed for these strains may confer a selective advantage in winemaking conditions. To test this hypothesis, we evaluated the relative selective advantage of representatives of the three main B. bruxellensis genetic groups in presence of SO2. As a proof-of-concept and using recently developed transformation cassettes, we compared strains under different SO2 concentrations using pairwise competitive fitness experiments. Our results showed that AWRI1499 is specifically adapted to environments with high SO2 concentrations compared to other B. bruxellensis wine strains, indicating a potential correlation between allotriploidisation origin and environmental adaptation in this species. Additionally, our findings suggest different types of competition between strains, such as coexistence and exclusion, revealing new insights on B. bruxellensis interactions at intraspecies level.

Measures to improve wine malolactic fermentation.

This review focuses on the considerable amount of research that has been directed towards the improvement of efficiency and reliability of malolactic fermentation (MLF), which is important in winemaking. From this large body of work, it is clear that reliable MLF is essential for process efficiency and prevention of spoilage in the final product. Impediments to successful MLF in wine, the impact of grape and wine ecology and how this may affect MLF outcome are discussed. Further focus is given to how MLF success may be enhanced, via alternative inoculation strategies, MLF progress sensing technologies and the use of different bacterial species. An update of how this information may be used to enhance and improve sensory outcomes through metabolite production during MLF and suggestions for future research priorities for the field are also provided.

Genomic insights into the metabolism of 'Candidatus Defluviicoccus seviourii', a member of Defluviicoccus cluster III abundant in industrial activated sludge.

Filamentous cluster III Defluviicoccus (DF3) are known to proliferate and cause bulking issues in industrial wastewater treatment plants. Members of the genus Defluviicoccus are also known to exhibit the glycogen accumulating organism (GAO) phenotype, which is suggested to be detrimental to enhanced biological phosphorus removal (EBPR). Despite the reported negative impact members of the DF3 have on activated sludge wastewater treatment systems, limited research has focused on understanding the physiological traits that allow them to compete in these environments. In this study, a near complete genome of an abundant filamentous DF3 named 'Candidatus Defluviicoccus seviourii' was obtained from a full-scale sequencing batch reactor (SBR) treating winery wastewater. Annotation of the 'Ca. D. seviourii' genome revealed interesting metabolic features that help to understand the abundance of this microorganism in industrial wastewater treatment plants. Their potential for the storage of polyhydroxyalkanoates (PHA) is suggested to favour these organisms with the intermittent availability of carbon in these systems. An ability to fix nitrogen and take up urea may provide them with an additional advantage with the characteristically high carbon to nitrogen content of industrial waste. The genome and preliminary findings of this study provide a foundation for further research into these biotechnologically relevant organisms.

Mass Transfer of Anthocyanins during Extraction from Pre-Fermentative Grape Solids under Simulated Fermentation Conditions: Effect of Convective Conditions.

The colour of red wine is largely determined by the concentration of anthocyanins that are extracted from grape skins during fermentation. Because colour is a key parameter in determining the overall quality of the finished product, understanding the effect of processing variables on anthocyanin extraction is critical for producing a red wine with the desired sensorial characteristics. In this study, the effect of convective conditions (natural and forced) on the mass transfer properties of malvidin-3-glucoside (M3G) from pre-fermentative grape solids was explored at various liquid phase conditions representing stages of fermentation. A mathematical model that separates solid and liquid phase mass transfer parameters was applied to experimental extraction curves, and in all cases, provided a coefficient of determination exceeding 0.97. Calculated mass transfer coefficients indicated that under forced convective conditions, the extraction process was controlled by internal diffusion whereas under natural convection, both internal diffusion and liquid-phase mass transfer were relevant in determining the overall extraction rate. Predictive simulations of M3G extraction during active fermentation were accomplished by incorporating the current results with a previously developed fermentation model, providing insight into the effect of a dynamic liquid phase on anthocyanin extraction.

Oenological traits of Lachancea thermotolerans show signs of domestication and allopatric differentiation.

The yeast Lachancea thermotolerans (previously Kluyveromyces thermotolerans) is a species of large, yet underexplored, oenological potential. This study delivers comprehensive oenological phenomes of 94 L. thermotolerans strains obtained from diverse ecological niches worldwide, classified in nine genetic groups based on their pre-determined microsatellite genotypes. The strains and the genetic groups were compared for their alcoholic fermentation performance, production of primary and secondary metabolites and pH modulation in Chardonnay grape juice fermentations. The common oenological features of L. thermotolerans strains were their glucophilic character, relatively extensive fermentation ability, low production of acetic acid and the formation of lactic acid, which significantly affected the pH of the wines. An untargeted analysis of volatile compounds, used for the first time in a population-scale phenotyping of a non-Saccharomyces yeast, revealed that 58 out of 90 volatiles were affected at an L. thermotolerans strain level. Besides the remarkable extent of intra-specific diversity, our results confirmed the distinct phenotypic performance of L. thermotolerans genetic groups. Together, these observations provide further support for the occurrence of domestication events and allopatric differentiation in L. thermotolerans population.

Modelling the Mass Transfer Process of Malvidin-3-Glucoside during Simulated Extraction from Fresh Grape Solids under Wine-Like Conditions.

Extraction of grape components is a key consideration for red winemaking. The impact of changing process variables on mass transfer properties of anthocyanins from fresh pre-fermentative red grape solids under forced convective conditions was explored using the dominant red grape anthocyanin, malvidin-3-glucoside (M3G) as a model solute. A two level full factorial design was implemented to investigate effects of temperature, sugar and ethanol on mass transfer properties. Factor levels were chosen to simulate conditions found at various points during the maceration and fermentation steps of the red winemaking process. A rigorous mathematical model was developed and applied to experimental extraction curves, allowing the separation of mass transport properties in liquid and solid phases in a wine-like system, for the first time. In all cases, the coefficient of determination exceeded 0.92, indicating good agreement between experimental and mathematically-solved M3G concentrations. For the conditions studied, internal mass transfer was found to limit M3G extraction and changes to the liquid phase composition and temperature influence the distribution constant. Surface response models of mass transfer parameters were developed to allow future simulations of fermentation scenarios aimed at maximising the extraction potential of M3G.

Genomic and in Situ Analyses Reveal the Micropruina spp. as Abundant Fermentative Glycogen Accumulating Organisms in Enhanced Biological Phosphorus Removal Systems.

Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the activity of polyphosphate accumulating organisms (PAOs). However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the Micropruina spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on Micropruina glycogenica str. Lg2T and compared to the in situ physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The Micropruina spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus in situ, but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal - as they compete for substrates with the Tetrasphaera PAO and stored carbon is not made available to the "Candidatus Accumulibacter" PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of potential carbon storage by fermentative organisms.

Genome Sequence of Australian Indigenous Wine Yeast Torulaspora delbrueckii COFT1 Using Nanopore Sequencing.

Here, we report the first sequenced genome of an indigenous Australian wine isolate of Torulaspora delbrueckii using the Oxford Nanopore MinION and Illumina HiSeq sequencing platforms. The genome size is 9.4 Mb and contains 4,831 genes.

Brettanomyces bruxellensis population survey reveals a diploid-triploid complex structured according to substrate of isolation and geographical distribution.

Brettanomyces bruxellensis is a unicellular fungus of increasing industrial and scientific interest over the past 15 years. Previous studies revealed high genotypic diversity amongst B. bruxellensis strains as well as strain-dependent phenotypic characteristics. Genomic assemblies revealed that some strains harbour triploid genomes and based upon prior genotyping it was inferred that a triploid population was widely dispersed across Australian wine regions. We performed an intraspecific diversity genotypic survey of 1488 B. bruxellensis isolates from 29 countries, 5 continents and 9 different fermentation niches. Using microsatellite analysis in combination with different statistical approaches, we demonstrate that the studied population is structured according to ploidy level, substrate of isolation and geographical origin of the strains, underlying the relative importance of each factor. We found that geographical origin has a different contribution to the population structure according to the substrate of origin, suggesting an anthropic influence on the spatial biodiversity of this microorganism of industrial interest. The observed clustering was correlated to variable stress response, as strains from different groups displayed variation in tolerance to the wine preservative sulfur dioxide (SO2). The potential contribution of the triploid state for adaptation to industrial fermentations and dissemination of the species B. bruxellensis is discussed.

Directed evolution of Oenococcus oeni strains for more efficient malolactic fermentation in a multi-stressor wine environment.

High concentrations of ethanol, low pH, the presence of sulfur dioxide and some polyphenols have been reported to inhibit Oenococcus oeni growth, thereby negatively affecting malolactic fermentation (MLF) of wine. In order to generate superior O. oeni strains that can conduct more efficient MLF, despite these multiple stressors, a continuous culture approach was designed to directly evolve an existing ethanol tolerant O. oeni strain, A90. The strain was grown for ∼350 generations in a red wine-like environment with increasing levels of stressors. Three strains were selected from screening experiments based on their completion of fermentation in a synthetic wine/wine blend with 15.1% (v/v) ethanol, 26 mg/L SO2 at pH 3.35 within 160 h, while the parent strain fermented no more than two thirds of l-malic acid in this medium. These superior strains also fermented faster and/or had a larger population in four different wines. A reduced or equivalent amount of the undesirable volatile, acetic acid, was produced by the optimised strains compared to a commercial strain in Mouvedre and Merlot wines. These findings demonstrate the feasibility of using directed evolution as a tool to generate more efficient MLF starters tailored for wines with multiple stressors.

Disruption of the cell wall integrity gene ECM33 results in improved fermentation by wine yeast.

Severe oenological conditions, such as limited assimilable nitrogen and high sugar contents restrict yeast's ability to successfully complete fermentation. In the absence of a comprehensive commercially available deletion collection in a wine yeast background, a screening approach was applied to a transposon library in a wine yeast derivative to identify clones with superior fermentation performance. Five candidate genes, when disrupted by Ty insertion, were identified as enabling yeast to efficiently complete a model oenological fermentation with limited nitrogen availability. Analogous single gene disruptions were subsequently constructed in the haploid wine yeast strain C911D, and the performance of these during fermentation was analysed. Deletion of ECM33 resulted in the shortest fermentation (up to 31% reduction) in both synthetic medium and grape juice. Interestingly, no significant differences were found in nitrogen utilization, cell viability or biomass yield between ∆ecm33 and the wild type. ∆ecm33 did, however, display growth hypersensitivity to the dyes Calcofluor White and Congo Red, suggesting a link to cell wall integrity. Transcriptional profiling of ∆ecm33 during fermentation demonstrated the up-regulation of SLT2 and HOG1, encoding mitogen activated protein kinases involved in the cell wall integrity (CWI) and high osmolarity glycerol (HOG) pathways, respectively. CHS3 a major chitin synthase gene was also found to be upregulated, and the transcript abundance of key genes of central nitrogen metabolism, GLN1, GLT1, GDH1 and GDH2 in mutant ∆ecm33 were also altered. The findings highlight the complexity of the robust fermentation phenotype and provide clues for further improvement of industrial strains.

Application of directed evolution to develop ethanol tolerant Oenococcus oeni for more efficient malolactic fermentation.

Malolactic fermentation (MLF) is an important step in winemaking, which can be notoriously unreliable due to the fastidious nature of Oenococcus oeni. This study aimed to use directed evolution (DE) to produce a more robust strain of O. oeni having the ability to withstand high ethanol concentrations. DE involves an organism mutating and potentially adapting to a high stress environment over the course of extended cultivation. A continuous culture of O. oeni was established and exposed to progressively increasing ethanol content such that after approximately 330 generations, an isolate from this culture was able to complete MLF in high ethanol content medium earlier than its parent. The ethanol tolerance of a single isolate, A90, was tested to confirm the phenotype and its fermentation performance in wine. In order to investigate the genotypic differences in the evolved strain that led to the ethanol tolerance phenotype, the relative expression of a number of known stress response genes was compared between SB3 and A90. Notably, there was increase in hsp18 expression in 20% (v/v) ethanol by both strains with A90 exhibiting a higher degree of expression. This study is the first to use directed evolution for O. oeni strain improvement and confirms that this technique can be used successfully for the development of new candidate strains for the wine industry. This study also adds to the current knowledge on the genetic basis of ethanol tolerance in this bacterium.

Managing the excessive proliferation of glycogen accumulating organisms in industrial activated sludge by nitrogen supplementation: A FISH-NanoSIMS approach.

Defluviicoccus vanus-related glycogen accumulating organisms (GAO) regularly proliferate in industrial wastewater treatment plants handling high carbon but nitrogen deficient wastes. When GAO dominate, they are associated with poor performance, characterised by slow settling biomass and turbid effluents. Although their ecophysiology has been studied thoroughly in domestic waste treatment plants, little attention has been paid to them in aerobic industrial systems. In this study, the effect of nitrogen addition on GAO carbon metabolism was investigated during an 8h cycle. Activated sludge dominated by GAO from a winery wastewater sequencing batch reactor was incubated under different carbon to nitrogen (COD:N) ratios (100:1, 60:1 and 20:1) using 13C - acetate and 15N - urea. GAO cell assimilation was quantified using FISH-NanoSIMS. The activated sludge community was assessed by 16S rRNA gene profiling, DNA and storage polymer production. Carbon and nitrogen quantification at the cellular level by NanoSIMS revealed that low (COD:N of 100:1) or null nitrogen concentrations enhanced GAO carbon uptake. COD:N ratios of 60:1 and 20:1 reduced GAO carbon uptake and promoted whole microbial community DNA production. Nitrogen dosing at COD:N ratios of 60:1 or higher was demonstrated as feasible strategy for controlling the excessive GAO growth in high COD waste treatment plants.

Remediation of Thiothrix spp. associated bulking problems by raw wastewater feeding: A full-scale experience.

An industrial wastewater treatment plant (WWTP) in Australia has long suffered from bulking problems associated with the proliferation of Thiothrix spp. The WWTP consists of a covered anaerobic lagoon (CAL) followed by a sequencing batch reactor (SBR). The CAL functions as both an anaerobic digester and surge lagoon for the irregular flow of wastewater generated from the production of seasonal products. Chemical analysis of the raw influent showed it was composed of a mixture of organic acids, phenols and alcohols. The CAL effluent was characterised by high acetic acid and phenolic concentrations. An attempt was made to manipulate the SBR microbial community to improve settling by direct feeding small volumes of raw influent into the SBR. After raw feeding, the plant ceased bulking as the settled sludge volume reduced from 930 to 200mLL-1. 16S rRNA gene profiling and biovolumes of SBR samples revealed major changes in the microbial community. The Thiothrix spp. population decreased from 36.8% to 0.2%, and Zoogloea spp. dominated all samples after raw feeding. Therefore, direct feeding is proposed as a control method for industrial plants with surge/anaerobic lagoons in order to manage the bulking problems caused by Thiothrix spp. in downstream SBRs.